In FMDV type C, amino acid substitutions seen in mutants that are resistant to neutralization by monoclonal antibodies (MAbs) map to predominantly surface-oriented residues with solvent-accessible side-chains not involved in interactions with other amino acids, whereas residues which are accessible but not substituted are found to be more frequently involved in protein-protein interactions. This provides a molecular interpretation for the repeated isolation of the same amino acid substitutions in MAb-resistant variants, an observation frequently made with RNA viruses. This first comparison of two FMDV serotypes shows how subtle changes at antigenic sites are sufficient to cause large changes in antigenic specificity between serotypes.

Foot-and-mouth disease virus (FMDV) enters cells by attaching to cellular receptor molecules of the integrin family, one of which has been identified as the RGD-binding integrin ␣v␤3. Here we report that, in addition to an integrin binding site, type O strains of FMDV share with natural ligands of ␣v␤3 (i.e., vitronectin and fibronectin) a specific affinity for heparin and that binding to the cellular form of this sulfated glycan, heparan sulfate, is required for efficient infection of cells in culture. Binding of the virus to paraformaldehyde-fixed cells was powerfully inhibited by agents such as heparin, that compete with heparan sulfate or by agents that compete for heparan sulfate (platelet factor 4) or that inactivate it (heparinase). Neither chondroitin sulfate, a structurally related component of the extracellular matrix, nor dextran sulfate appreciably inhibited binding. The functional importance of heparan sulfate binding was demonstrated by the facts that (i) infection of live cells by FMDV could also be blocked specifically by heparin, albeit at a much higher concentration of inhibitor; (ii) pretreatment of cells with heparinase reduced the number of plaques formed compared with that for untreated cells; and (iii) mutant cell lines deficient in heparan sulfate expression were unable to support plaque formation by FMDV, even though they remained equally susceptible to another picornavirus, bovine enterovirus. The results show that entry of type O FMDV into cells is a complex process and suggest that the initial contact with the cell surface is made through heparan sulfate.

The GH loop of VP1 is flexible in three serotypes of FMDV, suggesting that flexibility is important in both antigenic variability and structural communication with other regions of the virus capsid. Our results illustrate two instances of the propagation of structural perturbations across the virion surface: the change in the VP3 GH loop caused by the VP1 GH loop and the Glu82-->Gly change in VP2 which we believe perturbs the GH loop of VP1. In the latter case, the amplification of the sequence changes leads to differences, between the monolayer- and suspension-cell-adapted viruses, in host-cell interactions and antigenicity.

The integrin alpha(v)beta3 has been shown to act as the receptor for internalization of foot-and-mouth disease virus (FMDV) (A12), with attachment being through a highly conserved RGD motif located on the G-H loop of viral capsid protein VP1. In addition, however, we have recently shown that efficient infection of culture-grown cells by FMDV (O1BFS) requires binding to cell surface heparan sulfate. In this study, we have used a solid-phase receptor binding assay to characterize the binding by FMDV to purified alpha(v)beta3 in the absence of heparan sulfate and other cell surface components. In this assay, FMDV (O1BFS) successfully replicated authentic ligand binding by cellular alpha(v)beta3 in terms of its high affinity, dependence on divalent cations, and activation by manganese ions. Virus binding to this preparation of alpha(v)beta3 was exquisitely sensitive to competition by short RGD-containing peptides (50% inhibition at < 10(-8) M peptide), and this inhibition was highly sequence specific, with the equivalent RGE peptide being at least 10(4) fold less effective as a competitor. Representative viruses of the other six serotypes of FMDV bound to alpha(v)beta3 in a similar RGD-specific manner, although significant differences in sensitivity to RGD peptides suggest that the affinity of the different FMDV serotypes for alpha(v)beta3 is influenced, in part, by the variable amino acid residues in the VP1 G-H loop on either side of the RGD.

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